A novel pathway of levodopa metabolism by commensal Bifidobacteria

The gold-standard treatment for Parkinson’s disease is levodopa (L-DOPA), which is taken orally and absorbed intestinally. L-DOPA must reach the brain intact to exert its clinical effect; peripheral metabolism by host and microbial enzymes is a clinical management issue. The gut microbiota is altered in PD, with one consistent and unexplained observation being an increase in Bifidobacterium abundance among patients. Recently, certain Bifidobacterium species were shown to have the ability to metabolize L-tyrosine, an L-DOPA structural analog. Using both clinical cohort data and in vitro experimentation, we investigated the potential for commensal Bifidobacteria to metabolize this drug. In PD patients, Bifidobacterium abundance was positively correlated with L-DOPA dose and negatively with serum tyrosine concentration. In vitro experiments revealed that certain species, including B. bifidum, B. breve, and B. longum, were able to metabolize this drug via deamination followed by reduction to the compound 3,4-dihydroxyphenyl lactic acid (DHPLA) using existing tyrosine-metabolising genes. DHPLA appears to be a waste product generated during regeneration of NAD +. This metabolism occurs at low levels in rich medium, but is significantly upregulated in nutrient-limited minimal medium. Discovery of this novel metabolism of L-DOPA to DHPLA by a common commensal may help inform medication management in PD.

concentration that reached circulation was inversely proportional with TDC gene abundance in their guts 6 .Importantly, carbidopa did not inhibit these bacterial enzymes 6 .A second study from the same year made a similar discovery related to TDC enzymes in Enterococcus, and found that the bacterium Eggerthella lenta can sequentially dehydroxylate dopamine to tyramine 7 .The authors also showed that the tyrosine analog (S)-αfluoromethyltyrosine can inhibit the bacterial TDC enzymes and prevent L-DOPA decarboxylation, demonstrating the first proof of principle that inhibition of bacterial metabolism may be another tool to preserve L-DOPA's clinical efficacy.
Although decarboxylation has been the primary focus, other metabolic pathways may exist among the myriad enzymes encoded by the gut microbiota.Given the above TDC findings, and since tyrosine and L-DOPA differ in structure by only a single hydroxy group, existing tyrosine metabolism enzymes are the most likely sources of potential L-DOPA metabolism.Tyrosine can also be metabolized to 4-hydroxyphenyl lactic acid (HPLA) through a two-step reaction involving deamination by an aromatic amino acid aminotransferase (AAT) followed by reduction by an aromatic amino acid lactate dehydrogenase (ALDH).Lactate dehydrogenases are common among pathogens and environmental bacteria, but rarer among commensal gut microbes.However, it was recently found that certain Bifidobacterium species possess a previously unrecognized ALDH, and can produce lactic acids from aromatic amino acids including tyrosine 8 .This is intriguing as Bifidobacterium is one of the primary genera consistently seen at higher relative abundance in PD [9][10][11] , including in our own study 12 .Analogous to the ability of Enterococcus TDCs to convert L-DOPA to dopamine, we hypothesized that Bifidobacteria can use native tyrosine-metabolism enzymes to convert L-DOPA to its equivalent lactic acid, 3,4-dihydroxyphenyl lactic acid (DHPLA) (Fig. 1).

Participant recruitment and data collection
The participant cohort used in this study was recently described by our group 12 , including extensive methods on clinical data collection, fecal 16S microbiota analysis, and serum metabolomics analysis.Briefly, 197 PD patients and 103 controls were recruited from the Pacific Parkinson's Research Centre, University of British Columbia, between January 2017 and September 2019.During a study visit, a wide range of demographic and clinical data were collected, including L-DOPA dose.Participants were sent home with a fecal collection kit (OMNIgeneGUT Kit, DNA Genotek) and instructed to provide a fecal sample and mail it back to the centre at the next available opportunity; this kit contains a buffer which halts microbial growth and provides a snapshot of the microbiota at the moment of collection.Gut microbiota composition was analyzed using 16S sequencing of the V4 region, as described 12 .Notably, participant exclusion criteria included recent antibiotic and probiotic use, therefore any Bifidobacterium detected in their microbiota was expected to be stably colonized and not a result of recent probiotic ingestion.A small subset of patients from this study provided a fresh fecal sample on site during their visit, which was collected into a 50 mL conical tube and immediately flash frozen in liquid nitrogen then stored in a − 70 °C freezer.Isolated colonies used in this study originated from these flash-frozen samples.For methodological consistency, these participants also provided a preserved sample (OMNIgene GUT Kit) for use in 16S analysis.During the clinical visit, a blood sample was collected and untargeted metabolomics were performed on serum samples from 75 PD patients and 50 controls 12 .
This study was approved by the UBC Clinical Research Ethics Board (CREB) and conformed to all relevant CREB guidelines.All participants provided written informed consent.The study was performed in accordance with the Declaration of Helsinki.

Isolation of Bifidobacterium strains from PD fecal samples
On the day of isolation, the flash-frozen tube containing the freshly collected fecal sample was taken on ice into an anaerobic chamber, and a small amount of fecal material was added to a 2 mL Eppendorf tube containing 1 mL PBS with 0.1% cysteine.The sample was left on ice in the chamber for 30 min, then gently inverted until the sample appeared relatively homogenous, and larger particles were left to settle for 5 min.100 µL was then plated onto de-gassed De Man, Rogosa and Sharpe plates with 0.1% cysteine (MRS + C) using glass bead spread plating.The plates were incubated at 37 °C in the anaerobic chamber.
The following day, 20-30 colonies per plate were lightly touched with a sterile pipet tip and streaked onto new labelled MRS + C plates, then the tip was touched into a strip tube containing a pre-made 25 µL PCR mix (Q5 Hot Start High-Fidelity 2X Master Mix, NEB M0494L) and 8F/926R 16S primers (F: AGA GTT TGA TCC TGG CTC AG, R: CCG TCA ATT CCT TTR AGT TT).The newly streaked plates were returned to the incubator, and the PCR was run with the following conditions: 98 °C for 5 min, followed by 35 cycles of 98 °C for 20 s, 55 °C for 15 s, 72 °C for 45 s, then 72 °C for 10 min and a 4 °C hold.The reactions were cleaned using GeneJET PCR Purification kits (ThermoFisher K0701) and sent for Sanger sequencing (GeneWiz) using the 16S-8F primer.The resulting 600-700 bp sequences were interrogated using NCBI BLAST.Bacteria of interest were re-streaked onto another new plate, re-sequenced to confirm their identity, then grown up in 5 mL of liquid MRS + C broth from which glycerol stocks were made.Isolated bacteria were named using the convention "Genus species_##" where the identity was determined by Sanger sequencing of the 16S gene and the "##" refers to the anonymized patient ID that they were derived from.

Screening for tyrosine decarboxylase (TDC) activity
Bacterial isolates were screened for TDC activity using decarboxylation medium agar plates 13 containing basal nutrient sources, with or without 1% tyrosine.Medium composition is listed in Supplementary Table 1.The pH indicator bromocresol purple changes colour from yellow to purple if the more acidic amino acid (tyrosine) is converted to the more alkaline biogenic amine (tyramine).Bacteria were plated and grown anaerobically for 48 h at 37 °C.A colour change from yellow to purple on tyrosine plates, accompanied by no colour change on non-tyrosine control plates, indicated TDC activity.

Screening for tyrosine metabolism to HPLA
Bacteria were screened for their ability to convert tyrosine to 4-hydroxyphenyl lactic acid (HPLA) using mass spectrometry (MS).Bacteria were plated on MRS + C plates and individual colonies were transferred to 5 mL MRS + C broth overnight.Cultures were diluted to OD 600 = 0.01 in fresh MRS + C medium containing 0.01% pyridoxal 5'-phosphate (P5P), in a final volume of 1.2 mL in 2 mL snap-cap Eppendorf tubes.Tubes were anaerobically incubated for 48 h in a 37 °C shaking incubator (Eppendorf ThermoMixer C).Cultures were then spun at 16,000 × g at 4 °C for 10 min, and the clarified supernatant was aliquoted to new tubes and frozen at − 70 °C.
Metabolite extraction was performed as follows: samples were thawed on ice and spun at 16,000 × g at 4 °C for 5 min.80 µL of supernatant was added to tubes containing 320 µL acetonitrile + 0.1% formic acid.Samples were vortexed for 10 s then incubated at − 20 °C for 10 min, during which two distinct phases formed: a bottom turbid phase and a top translucent solvent phase.Samples were spun at 16,000 × g at 4 °C for 10 min to further separate the phases, and then 50 µL of the top phase was added to a tube containing 50 µL of H2O + 0.1% formic acid and 200 ng/µL deuterated L-DOPA-d 3 (MS internal standard, Cayman Chemicals 22089).The final sample was therefore a 1:10 dilution of the original, in 50% acetonitrile: 50% H2O + 0.1% formic acid, with 100 ng/µL internal standard.Metabolite extractions were prepared immediately prior to MS runs.

Mass spectrometry parameters
An LC-MS/MS system equipped with an Agilent Technologies 1200 high-performance liquid chromatography (HPLC) instrument, an Agilent 6460 triple quadrupole (QQQ) mass spectrometer, and a reversed-phase Nucleosil 100 C18 5 µm particle size column (Supelco) was used for MS runs.MS was conducted in positive and negative ion mode with an electrospray ionization voltage of 3500 V with 50 psi nebulizer gas at a temperature of 350 °C.Sample injection volume was 5 µL.LC separation was performed using mobile phases A (0.1% formic acid, 3% acetonitrile, 97% H 2 O) and B (0.1% formic acid, 90% acetonitrile, 10% H 2 O), at a flow rate of 400 µL/ min.The gradient program began with 15% B, increased linearly to 70% B over 7 min, was held for 1 min, then returned to 15% B over 1 min and was held for 3 min.A collision energy of 10 V was used for multiple-reaction monitoring.Data were analyzed using Mass Hunter Qualitative Analysis B.06.00 software (Agilent Technologies).
Positive/negative ion mode was selected according to the best ionization efficiency: dopamine, L-DOPA and L-DOPA-d 3 were run in positive; and HPLA and DHPLA were run in negative mode.Metabolite identification and quantification were carried out based on the retention time and mass fragmentation patterns compared with analytical standards.Six-point calibration curves were prepared, combining L-DOPA, dopamine, DHPLA, and HPLA together at final concentrations from 4000 to 10 ppb, all with 100 ppb of L-DOPA-d 3 as an internal standard.The R 2 for linearity was > 0.99 for all compounds.Metabolite concentrations were calculated by interpolating against calibration curves of individual compounds.LC-MS/MS instrument settings are available in Supplementary Table 2.

Enzymatic assays with cell-free extracts
Cell-free extracts (CFEs) for enzymatic assays were prepared based on an existing protocol 14 .Bacteria were grown for 16 h in 15 mL MRS + C broth, then spun for 15 min at 4800 × g at 4 °C.The supernatant was discarded and pellets were washed twice with 10 mL PBS using the same spin parameters.After the second wash, pellets were resuspended in 2 mL PBS and cells were lysed by sonication with a Fisher 550 Sonic Dismembrator on ice: [15 s on/15 s off] for 5 min total time at 20 kHz speed.Lysates were spun as above and the enzyme-containing supernatants were aliquoted and frozen at − 70 °C.Two 96-well-plate-based enzymatic assays were performed using these CFEs, as described 14 .First, aromatic amino acid aminotransferase (AAT) activity was assayed by monitoring conversion of tyrosine/L-DOPA to HPPA/DHPPA, respectively, by transfer of an amino group onto α-ketoglutarate, which causes an increase in absorbance at A 305 .Second, aromatic amino acid lactate dehydrogenase (ALDH) activity was assayed by monitoring reduction of HPPA/DHPPA to HPLA/DHPLA, respectively, using NADH as a substrate and measuring the decrease in absorbance at A 340 as NADH is converted to NAD +.For both assays, reactions without substrate and without CFEs were included as controls.Absorbance was measured in two minute intervals using a BioTek Synergy H1 plate reader.

Development of a Bifidobacterium minimal media
A Bifidobacterium minimal medium (BMM) was developed based on existing literature 15 .This defined medium uses lactose as a sole carbon source and ammonium acetate as a sole nitrogen source.Importantly, it also contains pyruvate, which is oxidized by the bacteria to regenerate NAD + from NADH.Full medium composition is listed in Supplementary Table 3.This medium was prepared with and without 1% casamino acids (CAA) as many Bifidobacterium species are auxotrophs for certain amino acids.All Bifidobacterium strains were able to grow on BMM + CAA, but only B. breve grew without CAA, displaying no amino acid auxotrophy.As L-DOPA metabolism involves amino acid metabolism pathways, further work with this minimal medium focused on B. breve in order to exclude exogenous amino acids.

Statistical analysis
The difference in Bifidobacterium relative abundance between PD patients and controls was assessed using DESeq2 16 with multiple testing correction for all bacterial genera, as we previously described 12 .Demographic and clinical differences between patients with low versus high Bifidobacterium abundance were assessed using Mann-Whitney U tests for continuous variables and Fisher's exact tests for categorical variables.Associations between Bifidobacterium abundance and serum metabolites were tested using Spearman correlation.Production of L-DOPA metabolites by mass spectrometry and enzymatic activity assays were assessed qualitatively.The difference in B. breve growth in various media conditions was assessed using a one-way ANOVA with Tukey's multiple comparison test.

Results
Bifidobacterium abundance is elevated in PD patients, and is associated with higher L-DOPA dosage In our cohort of 197 PD patients and 103 controls, we observed a significantly higher relative abundance of Bifidobacterium among PD patients (Fig. 2A), consistent with numerous published studies 9 .In order to investigate potential clinical differences between PD patients with lower versus higher levels of Bifidobacterium, we divided the patient cohort into two groups based on a nominal threshold of 1% Bifidobacterium relative abundance in their gut microbiota (Table 1).These groups were well matched on basic demographics (age, sex, BMI).Those with Bifidobacterium > 1% had a longer disease duration (8 vs 6 years) and slightly higher disease severity (MDS-UPDRS 17 ) scores, especially MDS-UPDRS Part IV which queries motor fluctuations which are typically caused by reduced duration and reliability of anti-parkinsonian medication 18 .
The most striking difference between the groups was that patients with Bifidobacterium abundance greater than 1% had an average L-DOPA dose 252 mg (43.0%) higher than those with Bifidobacterium less than 1% (Mann-Whitney U test p < 0.001, Fig. 2B).There was also a positive correlation between numerical Bifidobacterium abundance and L-DOPA dose (R s = 0.24, p < 0.001, Fig. 2C) 19 .
Patients with higher Bifidobacterium also had a significantly higher rate of entacapone use.Entacapone is a catecholamine-O-methyltransferase (COMT) inhibitor which prevents degradation of L-DOPA by the enzyme COMT and prolongs its half-life in the body 19 .When excluding patients taking entacapone, the L-DOPA dosage difference between patients with Bifidobacterium abundance below and above 1% remained significant (585 mg vs. 774 mg, 32.3% difference, Mann-Whitney U test p = 0.025).
To further investigate the association between Bifidobacterium and L-DOPA, a correlational analysis was performed using serum metabolomics data 12 , correlating all putative metabolite concentrations with Bifidobacterium relative abundance.Interestingly, the strongest correlation between any metabolite and Bifidobacterium www.nature.com/scientificreports/abundance was annotated as L-tyrosine in the Human Metabolome Database 21 .Bifidobacterium and L-tyrosine concentration displayed a negative correlation (R s = − 0.44, p < 0.001, Fig. 2D).The positive correlation between Bifidobacteria and L-DOPA dose, coupled with the negative correlation between Bifidobacteria and serum tyrosine, is consistent with the idea that Bifidobacteria are able to metabolize tyrosine (leading to a negative correlation) and perhaps L-DOPA using analogous pathways (leading to a higher dosage requirement, and/or the higher doses creating a gut environment that confers some growth advantage to Bifidobacterium).

Bifidobacteria do not decarboxylate L-DOPA to dopamine
Decarboxylation medium plates revealed a lack of tyrosine decarboxylation activity among all Bifidobacterium isolates screened, with Enterococcus faecalis, a bacterium with known tyrosine decarboxylase ability 6 , serving as a positive control (Supplementary Fig. 1).As the decarboxylation medium plates may not have had suitable sensitivity for detecting very low amounts of dopamine production, we further interrogated this via mass spectrometry, where our detection limit was 62.5 ng/mL.All Bifidobacterium strains, and E. faecalis, were incubated with 1 mM L-DOPA for 48 h and production of dopamine was quantified by LC-MS/MS.E. faecalis produced 1 mM dopamine, representing ~ 100% decarboxylation of L-DOPA, while dopamine was undetectable in all Bifidobacterium samples.Therefore, the ability of Bifidobacteria to decarboxylate L-DOPA to dopamine was ruled out.

Tyrosine metabolism by Bifidobacteria
Reference genomes of all utilized Bifidobacterium species were searched for annotated lactate dehydrogenase genes using NCBI GenBank, and it was found-as also described by Laursen et al. 8 -that B. longum, B. bifidum, and B. breve contain a lactate dehydrogenase in close proximity to an aromatic amino acid aminotransferase in what may be a potential operon (Supplementary Fig. 2).To test the functionality of these genes, Bifidobacterium strains were screened for their ability to produce 4-hydroxyphenyl lactic acid (HPLA) from MRS-C media, which naturally contains tyrosine.It was found that B. bifidum, B. breve, and all strains of B. longum produced relatively high amounts of HPLA (Fig. 3A), while strains of B. adolescentis, B. animalis, B. stercoris, and E. faecalis did not.These data match the findings by Laursen et al. 8 .

Conversion of L-DOPA to the aromatic lactic acid DHPLA
Bacteria were then screened for their ability to convert L-DOPA to the equivalent aromatic lactic acid, 3,4-dihydroxyphenyl lactic acid (DHPLA), from MRS + C medium supplemented with 1 mM L-DOPA.There was high concordance between bacteria able to produce HPLA from tyrosine, and those able to generate DHPLA from L-DOPA (Fig. 3B).However, conversion rates to DHPLA were low, with only four isolates producing > 10 µM DHPLA which represents 1% conversion of the initial 1 mM L-DOPA.Importantly, there was no spontaneous generation of DHPLA in media without bacteria, nor with E. faecalis which served as a negative control.Therefore, it was concluded that several Bifidobacterium species possess this metabolic capacity, however in a nutrientrich medium, this reaction appears to exhibit limited activation, and/or L-DOPA is not the preferred substrate.To investigate the dynamics of this reaction, B. longum_08 was incubated with either 1 mM L-DOPA, 1 mM DHPPA, or 1 mM DHPLA; and L-DOPA and DHPLA were quantified after 48 h (Fig. 4A).In controls without bacteria, 100% of the starting compound was recovered, indicating no spontaneous breakdown.With bacteria + L-DOPA, B. longum converted ~ 2% into DHPLA, as previously seen.Interestingly, with bacteria + DHPPA, B. longum converted ~ 15% to DHPLA, and the remainder was actually converted back to L-DOPA.This indicates that the transamination reaction is skewed in the direction of making L-DOPA (i.e.making tyrosine under normal conditions) in rich medium.In the condition with bacteria + DHPLA, 100% DHPLA was recovered and L-DOPA was not detected, revealing that DHPLA is indeed a final metabolic end-product and suggesting that any generation of DHPLA is irreversible.This experiment was repeated with the remaining bacteria of interest, with similar results (Fig. 4B).Bacteria + L-DOPA converted ~ 1-3% of the L-DOPA to DHPLA, and when DHPPA was the starting compound, they variably converted between ~ 10-40% to DHPLA and the remainder back to L-DOPA.All bacteria incubated with DHPLA resulted in 100% DHPLA recovery.In conclusion, this demonstrates that the deamination reaction (L-DOPA → DHPPA) is the rate-limiting step in the conversion of L-DOPA to DHLPA and that it is balanced in the reverse direction in nutrient-rich medium, but that any L-DOPA that is converted to DHPLA is permanently lost as a waste product.

Enzymatic capability for L-DOPA metabolism
We next more closely investigated the kinetics of both the deamination (L-DOPA → DHPPA) and reduction (DHPPA → DHPLA) reactions using cell-free-extract (CFE) enzymatic assays.In addition to the Bifidobacterium isolates of interest, two environmental Lactobacilli (L.fermentum and L. plantarum) were used due to their known ability to produce high amounts of the tyrosine metabolite HPLA [22][23][24] .The first assay measured AAT activity,  which deaminates tyrosine to HPPA and L-DOPA to DHPPA.The two Lactobacilli did not have this capability; however, all four Bifidobacteria tested were able to perform this reaction (Fig. 5A,B).In each case, the reaction was faster with tyrosine than with L-DOPA, which is unsurprising as tyrosine is the natural substrate.
The second assay measured ALDH-mediated reduction of HPPA to HPLA and DHPPA to DHPLA.The two Lactobacilli, as expected, performed this reaction with both substrates extremely efficiently.The four Bifidobacteria also carried out the reaction with both substrates, albeit at a slower rate, and with higher background activity (i.e.other components in the CFE were also potential substrates) (Fig. 5C,D).For both enzymatic assays, negative controls without CFEs showed no background activity.

L-DOPA metabolism in minimal media
To investigate whether the metabolism of L-DOPA by Bifidobacterium is upregulated in a nutrient-limited environment, a defined minimal media (BMM) was employed.Of all strains, only B. breve was able to grow in this medium without exogenous amino acids and was thus the focus of these experiments.Growth in this media supplemented with 1 mM L-DOPA for 48 h resulted in 126.2 µM DHPLA production as measured by LC-MS/ MS, representing ~ 12% conversion, significantly higher than the ~ 1% observed in rich MRS + C medium.No DHPLA was produced in BMM without L-DOPA, ruling out spontaneous production of this molecule.
According to the proposed pathway of L-DOPA metabolism, the final step-conversion of DHPPA to the metabolic end-product DHPLA-is a redox reaction used by the bacteria to regenerate NAD + from NADH.In the minimal medium, this anaerobic homolactic fermentation reaction occurs via conversion of pyruvate to lactic acid.Removal of pyruvate from BMM resulted in complete inhibition of B. breve growth.When an equimolar amount of DHPPA was added to the media, growth was completely restored (Fig. 6), confirming that B. breve can use this L-DOPA metabolite for the essential function of NAD + regeneration.

Discussion
To provide dependable symptom relief, it is critical that L-DOPA reaches the brain intact.It has recently become clear that in addition to host peripheral metabolism, metabolism by gut bacteria may also be a concern 6,7 .This study describes a novel pathway of L-DOPA metabolism by certain Bifidobacterium species employing existing Here, significant background activity occurred without any substrate, however all bacteria performed this reaction above background levels as summarized in (D).In both assays, no reactions occurred without CFEs.tyrosine metabolism genes.In PD patients, we observed a negative correlation between Bifidobacterium and serum tyrosine.This is similar to the pattern seen in fecal samples from breastfed infants 8 , revealing strong evidence for tyrosine metabolism by these bacteria in vivo.A recent study also observed higher Bifidobacterium and lower concentrations of aromatic amino acids in PD fecal samples 25 .
Bifidobacteria were not able to decarboxylate L-DOPA to dopamine, a pathway recently observed in Enterococcus and Eggerthella species 6,7 ; therefore, we searched for alternative pathways to explain our clinical observations.Non-commensals-including pathogens and environmental bacteria-frequently encode aromatic amino acid aminotransferases [26][27][28] that are possibly able to deaminate L-DOPA to DHPPA, and lactate dehydrogenases 8,29,30 that could reduce DHPPA to DHPLA.Indeed, a recent study found that the soil bacterium Clostridium sporogenes can efficiently deaminate L-DOPA to DHPPA using an aminotransferase 31 .Our study describes the first observation of the complete metabolic pathway from L-DOPA to the aromatic lactic acid DHPLA among common gut commensals.
Compared to the ability of Enterococcus to metabolize L-DOPA, Bifidobacteria do so much less efficiently; Enterococcus has been shown to metabolize 100% of L-DOPA in culture to dopamine within 24h 6,7 (which we also observed), whereas strains of Bifidobacteria converted 1-3% to DHLPA in 48 h in rich media, and approximately 12% in nutrient-limited media.Importantly, however, the reduction of DHPPA to DHPLA appears to be irreversible, with DHPLA being a final end-product analogous to lactic acid generation by these bacteria during homolactic fermentation 32 .Therefore, any DHPLA generated becomes permanently unavailable for reconversion to L-DOPA.In minimal medium, which likely more closely resembles the nutrient-deprived conditions in the colon, substantially more L-DOPA is converted into the final lactic acid end-product by these bacteria.
The strong association we observed between higher Bifidobacterium abundance and entacapone use has been previously noted in other studies [33][34][35] .As a catechol-O-methyltransferase inhibitor, entacapone serves to prolong the half-life of L-DOPA in the body by preventing its degradation by COMT.This would likely increase the fraction of non-metabolized L-DOPA that reaches the colon, where it would become available for microbiota metabolism, thus potentially explaining the positive association between Bifidobacterium and entacapone.However, entacapone use has been shown to affect the abundance of other gut bacterial genera as well 33,35 , therefore it may have larger scale effects on the microbiome or on the overall gut environment that are not yet fully understood.
The observed positive correlation between Bifidobacterium abundance and L-DOPA dose in PD patients has two possible interpretations.The first is that patients with higher levels of Bifidobacterium require higher medication dose because their gut bacteria are metabolizing a significant portion and making it clinically unavailable.The second is that in patients with naturally higher medication doses (for clinical reasons), a growth advantage is conferred to Bifidobacterium as they are able to use residual L-DOPA for metabolic purposes.In light of the fairly modest metabolism of this drug, and the fact that the highest microbiota biomass and metabolic activity is in the colon 32 whereas L-DOPA is absorbed in the upper small intestine 1,3 , the second interpretation is more plausible.Approximately 10% of orally ingested L-DOPA is never absorbed into systemic circulation 31,36,37 , leaving ample substrate for bacterial metabolism in the lower GI tract.Thus, the administration of L-DOPA may provide a fitness advantage to microorganisms harboring this low-affinity transformation pathway.Interestingly, although many human cohort studies report higher levels of Bifidobacterium among PD patients, one large study of medication-naïve patients 38 and another of (also L-DOPA-naïve) individuals with idiopathic rapid eye movement sleep behavior disorder 39 -a condition with extremely high conversion to PD-both observed no differences in Bifidobacterium abundance between patients and controls.This perhaps suggests that it is indeed the initiation of L-DOPA administration that drives a subsequent increase in Bifidobacterium.This is further supported by the longer disease duration, worse disease severity, and increased entacapone use among PD patients with higher Bifidobacterium in our cohort; in other words, it is more likely that patients' disease advancement and the associated increase in L-DOPA dose (and use of L-DOPA-stabilizing drugs) drives subsequent Bifidobacterium flourishing, rather than higher Bifidobacterium abundance driving increased dose requirements.This is also borne out by the fact that most microbial metabolism (in the colon) is downstream of the site of clinically-relevant L-DOPA absorption (in the upper small intestine).Large studies specifically investigating changes in microbial composition pre-and post-L-DOPA initiation in newly diagnosed PD patients will further inform this hypothesis.
Lactobacillus is another genus that employs homolactic fermentation, and interestingly, this genus has also been reported to be higher in abundance in PD 10,11 .In our cohort, we observed very little of this genus overall (both in patients and controls) 12 , and during the isolation of Bifidobacterium strains, no Lactobacilli were found despite the fact that they grow well on MRS medium.Therefore, due to the lack of supporting clinical data and isolates, whether this pathway also exists in any commensal gut Lactobacilli was not explored; however, it is an intriguing possibility.
This study has several limitations.The human cohort data is associative, and no causality can be ascribedindeed, we offer two contrasting explanations for the positive correlation between Bifidobacterium abundance and L-DOPA dose among PD patients.Our microbiota data was based on 16S analysis which did not discriminate Bifidobacteria to the species level, therefore our results group the entire genus, although it is clear that only certain species can perform this reaction.The in vitro studies are limited by the fact that they are single-organism in nature.In reality, the microbiota is a complex community which involves competition, cross-feeding, and environmental conditions that cannot be replicated in a culture tube.
We urge caution against over-interpreting these results.As mentioned, the efficiency of this L-DOPA metabolism reaction in Bifidobacterium is quite low, reaching only ~ 1% in complete medium and only ~ 12% in minimal medium after 48 h.Indeed, in nutrient-rich medium, the aminotransferase reaction is actually balanced in the direction of regenerating L-DOPA from the intermediate pyruvic acid, implying that in certain contexts Bifidobacteria could actually protect the integrity of L-DOPA against bacteria that efficiently deaminate (but not reduce) it.Bifidobacterium-containing probiotics have shown benefits in many aspects that may be relevant to PD, including improving mood and gastrointestinal function [40][41][42][43] .Therefore, if PD patients report positive experiences from probiotic use, we suggest that the benefits likely outweigh the possible low-level metabolism of residual L-DOPA by these species and we do not suggest discontinuing probiotic administration based on the results of this study.
As a final note, beyond direct metabolism by gut bacteria, other factors related to gut health also impact the pharmaceutical efficacy of L-DOPA.Gastroparesis, slow colonic motility, and constipation can all negatively impact L-DOPA absorption and lead to unpredictable "OFF" periods 5 .Therefore, any interventions, microbial or otherwise, that restore normal gut health and function could have the added benefit of aiding with medication management and symptom relief in this population. www.nature.com/scientificreports/

Figure 1 .
Figure 1.Proposed metabolism pathways.L-DOPA metabolism (right) and analogous pathways of tyrosine metabolism (left).The only difference between the two molecules is a hydroxy group on the unmetabolized aromatic ring, highlighted by a red star.TOP: Decarboxylation of tyrosine to tyramine (left) and of L-DOPA to dopamine (right), a pathway known to exist in Enterococcus spp.BOTTOM: Deamination and reduction of tyrosine to HPLA (left), a pathway recently shown to exist in certain Bifidobacterium spp; and the proposed analogous pathway of L-DOPA conversion to DHPLA (right).

Figure 2 .
Figure 2. Human clinical data.(A) PD patients had a higher relative abundance of Bifidobacterium than controls.(B) PD patients with Bifidobacterium relative abundance > 1% had a significantly higher L-DOPA dose.(C) Bifidobacterium abundance (center-log ratio transformed) was positively correlated with L-DOPA dose in PD patients.(D) Bifidobacterium relative abundance was negatively correlated with serum tyrosine concentration.

Figure 3 .
Figure 3. Production of HPLA and DHPLA by Bifidobacterium.(A) Production of the tyrosine metabolite HPLA by various strains of Bifidobacterium.(B) Production of the L-DOPA metabolite DHPLA by the same bacteria.

Figure 4 .
Figure 4. Bifidobacteria prefer to re-aminate DHPPA in rich medium.Bacteria were cultured in MRS + C medium with 1 mM of L-DOPA, the intermediate DHPPA, or the end-product DHPLA.(A) B. longum converted a small amount of L-DOPA to DHPLA.When incubated with DHPPA, it reduced ~ 15% to DHPLA and the remainder was re-aminated back to L-DOPA.When incubated with DHPLA, no further metabolism occurred.(B) This experiment was repeated using additional strains with similar results.

Figure 5 .
Figure 5. Cell-free extract (CFE) assays demonstrate L-DOPA metabolism potential.(A) Representative aminotransferase (AAT) assay with B. breve.With no substrate, no amino-group transfer to α-ketoglutarate (detected by an increase in A 305 ) occurs.With tyrosine, and L-DOPA at a slower rate, this reaction takes place.A 305 at time = 0 are normalized to 0. (B) Summarized results of the change in OD 305 after 60 min for all bacteria.(C) Representative lactate dehydrogenase (ALDH) assay for B. breve in which oxidation of NADH to NAD + causes a decrease in A 340 .Here, significant background activity occurred without any substrate, however all bacteria performed this reaction above background levels as summarized in (D).In both assays, no reactions occurred without CFEs.

Figure 6 .
Figure 6.Minimal medium replacement experiment.In minimal medium, pyruvate is essential for B. breve growth.The L-DOPA metabolite DHPPA is able to replace pyruvate and restore growth.Cultures were grown for 48 h.

Table 1 .
Demographics and clinical characteristics of PD patients with Bifidobacterium relative abundance in their gut microbiota below versus above 1%.Significant values are in bold.